Summary

Synthetic mRNAs can be injected to achieve transient gene expression even
for `non-model' organisms in which genetic approaches are not feasible. Here,
we have used this technique to express proteins that can serve as lineage
tracers or reporters of cellular events in embryos of the glossiphoniid leech
Helobdella robusta (phylum Annelida). As representatives of the
proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of
interest for developmental and evolutionary comparisons. Their embryos are
suitable for microinjection, but no genetic approaches are currently
available. We have injected segmentation stem cells (teloblasts) with mRNAs
encoding nuclear localized green fluorescent protein (nGFP) and its spectral
variants, and have used tandem injections of nGFP mRNA followed by antisense
morpholino oligomer (AS MO), to label single blast cell clones. These
techniques permit high resolution cell lineage tracing in living embryos. We
have applied them to the primary neurogenic (N) lineage, in which alternate
segmental founder cells (nf and ns blast cells) contribute distinct sets of
progeny to the segmental ganglia. The nf and ns blast cell clones exhibit
strikingly different cell division patterns: the increase in cell number
within the nf clone is roughly linear, while that in the ns clone is almost
exponential. To analyze spindle dynamics in the asymmetric divisions of
individual blast cells, we have injected teloblasts with mRNA encoding a
tau::GFP fusion protein. Our results show that the asymmetric divisions of n
blast cells result from a posterior shift of both the spindle within the cell
and the midbody within the mitotic spindle, with differential regulation of
these processes between nf and ns.

Events in the N lineage leading to gangliogenesis in H. robusta.
Cleavage yields an embryo with a posterior growth zone of five bilateral pairs
of segmentation stem cells (teloblasts); only the four pairs of ectoteloblasts
are shown here. Ignoring gastrulation, segments arise in anteroposterior
progression along the ventral midline by the stereotyped divisions of columns
of segmental founder cells (blast cell bandlets). In the N (and Q lineages),
clones of two successive blast cells, designated ns (red) and nf (blue)
contribute distinct sets of progeny to each segment. Because blast cells of
each type execute a stereotyped pattern of cell division relative to the time
of their birth from the parent teloblast, events in the generic nf and ns
lineages can be designated as occurring at a specific `clonal age' (cl.ag.),
as indicated on the time line on the left. Primary n blast cells divide
unequally, producing larger anterior (nf.a, dark blue; ns.a red) and smaller
posterior (nf.p, light blue; ns.p, pink) daughter cells (40 and 44 hours
cl.ag., respectively). Transverse fissures (arrows) separate nf.p clones
(light blue) from ns.a clones (red) dividing the bandlets into discrete
ganglionic primordia (85-95 hours cl.ag.). Anterior is upwards in all figures
unless otherwise noted.

Each teloblast lineage exhibits a specific pattern of blast cell division
patterns (Zackson, 1984;
Bissen and Weisblat, 1989). For
the N and Q lineages, this includes differences in the timing and symmetry of
the early divisions of alternating blast cells, designated as nf and ns, qf
and qs, respectively. An intriguing issue is whether these cells assume
distinct fates at birth or as a result of interactions within the n and q
bandlets. Segment-specific identities of blast cells in the M and O lineages
are assigned long before the primary blast cells divide
(Martindale and Shankland,
1990; Gleizer and Stent,
1993; Nardelli-Haefliger et
al., 1994), and results correlating the nf and ns fates with
subtle differences in the teloblast cell cycle support the grandparental mode
model (Bissen and Weisblat,
1987). The overt differences between the nf and ns blast cells in
the timing and symmetry of their initial mitoses provide a point of entry to
exploring how nf and ns fates are assigned.

Microinjection of fluorescent dextrans
(Gimlich and Braun, 1985) has
been the technique of choice for analyzing cell lineages in
Helobdella, but it becomes difficult to distinguish individual cells
in the marked clones as the cells increase in number and decrease in size,
especially when they lie adjacent to one another. More recently, diverse
applications employing fluorescent proteins (GFP, YFP, CFP and DsRed;
designated collectively here as XFPs) have been used to label specific cells
in vivo (Tsien, 1998;
Lippincott-Schwartz and Patterson,
2003). These fluorescent proteins can be fused to target genes,
allowing inferences to be drawn about their expression and localization in
live embryos of various sorts (Chalfie et
al., 1994; Lee and Luo,
1999; Amsterdam et al.,
1995; Feng et al.,
2000).

Here, we have injected mRNAs encoding various XFP constructs in a
combination of lineage tracing and reporter construct technologies, focusing
on the primary neurogenic (N teloblast) lineage in H. robusta. We
found that these mRNAs meet all the criteria for being microinjectable lineage
tracers. In particular, the spatially restricted fluorescence of nuclearly
localized XFP (nXFP) enabled us to extend our knowledge of the nf and ns blast
cell lineages and revealed dramatic differences between them. We have also
characterized the degree of asymmetry of the primary nf and ns blast cell
divisions by using nXFP fluorescence to measure the nuclear volumes of the
daughter cells. To follow the spindle dynamics of dividing nf and ns blast
cells, we injected mRNA encoding tau::GFP. The spindle dynamics in the
dividing ns and nf cells showed both similarities and differences to those in
the asymmetric divisions of the C. elegans zygote and
Drosophila neuroblasts.

Materials and methods

Embryos

Embryos were obtained from a laboratory-cultured strain provisionally
identified as an Austin (Texas) strain of Helobdella robusta
(Seaver and Shankland, 2000;
Kuo and Shankland, 2004).
Embryos were cultured in HL saline and maintained at 23°C. Staging and
cell nomenclature are as defined previously
(Weisblat and Huang, 2001) for
the holotypic strain of H. robusta collected in Sacramento
(California) (Shankland et al.,
1992), but the strains differ in the relative rates at which they
progress through certain stages (S.O.Z. and D.A.W., unpublished), and in the
sequences of various genes that have been studied (A. E. Bely, personal
communication). In Helobdella, segments arise from seven distinct
sublineages, founded by m, nf, ns, o, p, qf and qs blast cells (see
Fig. 1 for details).

Plasmid constructs, mRNA synthesis and mRNA injection

eGFP mRNA was transcribed in vitro from linearized pCS2P-eGFP-X/P plasmid.
Nuclear localized GFP and β-galactosidase (nGFP and nLacZ) and tau::GFP
mRNAs were transcribed from pCS2P-nls-eGFP, pCS2-nls-βgal and
pCS2-tau-GFP plasmids, respectively. To make nuclear localized versions of
other fluorescent proteins, plasmids pECFP-N1, pEYFP-N1 and pDsRed2-N1
(Clontech) were used as templates (details available on request). RNA was
injected from standard glass pipets treated to avoid RNase contamination
(details available upon request). The concentration of mRNAs in the needle are
0.4 μg/μl (GFP, nGFP, nCFP, nYFP, nRFP, tau::GFP) or 0.1 μg/μl
(nLacZ), either with or without 5 μg/μl rhodamine dextran amine (RDA,
Molecular Probes), the final concentration of mRNAs in the teloblast was
estimated to be 4 ng/μl or 1 ng/μl, as the injected volume is estimated
to be 1% of that of the N teloblast.

Morpholino injection

Antisense morpholino oligomer (AS MO, Gene Tools) complementary to the
start codon and seven downstream codons of nGFP mRNA, designated as AS-nGFP MO
(5′-CCTTACGCTTCTTCTTTGGAGCCAT-3′; anti-start codon
underlined), was injected at a concentration of 0.1 mM in the needle (1 μM
in the teloblast) if not otherwise indicated. AS-nGFP MO was used as a
4-mismatch control morpholino to nCFP and nYFP mRNA, which are similar in this
region (5′-ATGGCaCCgAAGAAGAAGaGgAAGG-3′; mismatches are
in lowercase and the start codon is underlined). The sequence of AS-nLacZ MO
is 5′-TACGCTTCTTCTTTGGAGCAGTCAT-3′ with the anti-start
codon underlined. Injections procedures were as for mRNA injection. At least
20 embryos were injected for each experimental time point and all experiments
were performed in triplicate at minimum.

Confocal image stacks of ns/nf clones and stage 9-10 ganglia were
deconvolved and reconstructed using Imaris 4.0 (Bitplane AG), or Volocity 3.0
(Improvision); nuclei were annotated and measured in Volocity. Epifluorescence
images were deconvolved (2-D), then reconstructed in Metamorph. Images were
exported as movies and single snapshots, and further processed with Photoshop
5.0 (Adobe) to prepare figures. Spindle images were processed and converted
into time-lapse movies using Metamorph.

In situ hybridization, X-gal staining and Hoechst staining

Antisense GFP probe was made using T7 MEGAscript kit (Ambion) and
hybridizations were as previously described
(Song et al., 2002). X-gal
staining was as previously described (Liu
et al., 1998), or as modified from a protocol in Xenopus
(Sive et al., 2000) (details
upon request). Embryos were counterstained and mounted for observations as
previously described (Shain et al.,
2000).

Results

Nuclear localized fluorescent proteins as lineage tracers

For cell lineage studies using fluorescently labeled dextrans or GFP, the
cytoplasmic distribution of the fluorescence makes it hard to distinguish
individual cells in the germinal bands and germinal plate, especially in
lineages where small cells are closely apposed
(Bissen and Weisblat, 1989;
Huang et al., 2002). To
circumvent these problems, we tested mRNA encoding nuclear localized GFP
(nGFP) as a cell lineage tracer in living embryos, and found that it meets the
three criteria required of any microinjected lineage tracer: (1) confinement
to the clone of the injected cell; (2) stability and detectability during
extended periods of development; and (3) not perturbing normal development.
This last criterion was of particular concern given the nuclear localization
of the nGFP; it was easy to imagine that it might perturb the normal
lineages.

N teloblasts in stage 7 embryos (∼33 hours AZD) were injected with RDA
and nGFP mRNA, After ∼48 hours of subsequent development, the pattern of
nGFP-labeled nuclei was as expected from previous studies
(Zackson, 1984;
Bissen and Weisblat, 1989) (not
shown). In other experiments, we allowed injected embryos to develop for 143
hours post-injection. As at earlier stages, the distribution of marked cells
was indistinguishable from when teloblasts were with injected with RDA only.
The nGFP was still readily detectable, and cell nuclei in the labeled clone
were observed with excellent resolution. We counted ∼100 nuclei in the
anteriormost labeled hemiganglion and the N-derived peripheral neurons (nz1-3)
were observed at their stereotyped positions
(Fig. 2A,B). In a third set of
embryos, both N and P teloblasts were injected with nGFP mRNA and examined at∼
100 hours post-injection; neural precursor cells had migrated medially
from the p bandlet (Fig. 2C) as
reported previously for Theromyzon
(Torrence and Stuart, 1986)
and Helobdella (Braun and Stent,
1989). Thus, nGFP showed excellent perdurance and did not
noticeably perturb development (Fig.
2D).

nXFPs as lineage tracers in Helobdella. (A) Photomontage made from
stacks of confocal images (processed by Imaris, Bitplane) showing a ventral
view (midline to left) of a stage 10 embryo, 140 hours after the left N
teloblast had been injected with nGFP mRNA. nGFP labeled nuclei occupy the
anterior and posterior lobes of N-derived ganglionic neurons and the three
peripheral neurons lying just outside the posterior (nf-derived) lobe
(arrows). (B) Higher magnification view of the boxed region in A. (C)
Epifluorescence images of the germinal plate of a late stage 8 embryo (ventral
midline towards the left) about 100 hours after the left N and P teloblasts
were injected with nGFP mRNA. The N and P lineages can be distinguished by a
difference in fluorescence intensity, reflecting differences in the amount of
mRNA injected into the parent teloblasts. The temporal gradient of development
is evident in the progress of migrating neurons (arrowheads) which originate
from a single cell at the medial edge of the p blast cell clone (arrow). (D)
Side view of an early stage 10 embryo, 150 hours after the left N teloblast
had been injected with nGFP mRNA shows an anterior (arrowhead) to posterior
gradient of GFP intensity. (E) Pseudocolored image (confocal stack) showing a
ventral view of a live mid stage 8 embryo, 83 hours after one N teloblast had
been injected at stage 7 with nCFP mRNA and the other with nYFP mRNA 3 hours
later (nCFP, purple; nYFP, green). Scale bar: 20 μm.

Spectral variants of GFP (collectively designated XFPs) are widely used
(Lippincott-Schwartz and Patterson,
2003). We made and tested several XFPs for use in multi-label
lineage tracing (see Materials and methods for details). Injection of mRNAs
for nCFP and nYFP produced strong, nuclear-localized fluorescence
(Fig. 2E), with the same
perdurance and benignity as nGFP; nRFP, however, showed a much shorter
half-life (data not shown), so the version of nRFP used here is not useful for
long term lineage tracing in Helobdella.

To further refine the mRNA injections technique in Helobdella, we
used it to test the efficacy of antisense knockdown reagents. RNAi did not
yield reproducible knockdowns (data not shown), but antisense morpholino
oligomer (AS MO) injections did. For this purpose, teloblasts were injected
with nGFP mRNA and then after 3 hours, with AS MO complementary to 5′
sequence of nGFP mRNA (AS-nGFP MO; see Materials and methods for details). We
observed a significant and long-lasting knockdown of nGFP fluorescence in the
primary blast cells and their clones that received AS-nGFP MO, relative to the
cells produced prior to the second injection
(Fig. 3A,B); in similar
experiments, AS MO knockdown of nLacZ expression was also obtained (data not
shown). Using quantitative fluorescence measurements, we estimate a knockdown
of about 50% (Fig. 3C). No
knockdown occurred when the target mRNA bore a mismatched target sequence or
when the second injection comprised RDA only (data not shown).

AS MO knockdown of injected mRNA expression. (A,B) N teloblasts were
injected at stage 6 with nGFP mRNA, 3 hours later with a mixture of
fluorescent dextran (RDA) and antisense morpholino oligomer (AS MO), and then
imaged after another 46 and 98 hours. The left side of each panel shows the
combined RDA (red) and nGFP (green), and the right side shows nGFP only,
revealing the decrease in nGFP fluorescence in clones produced after the AS MO
injection. In these experiments, two or three blast cells were produced
between the first and second injections. (C) Enlarged view of the embryo shown
in A; fluorescence intensity was measured along the line shown crossing blast
cell nuclei produced before and after the AS MO injection and plotted on the
right. Scale bar: 10 μm.

Distribution and perdurance of injected mRNA and protein

We routinely observed a gradient of increasing GFP fluorescence within the
progeny of the injected teloblasts (Fig.
2A,D,E). The nGFP fluorescence must be determined by a complex
dynamic involving nGFP mRNA and protein. To examine the stability and
distribution of the injected mRNA, N teloblasts were injected with nGFP mRNA
in stage 7 embryos (33 hours AZD), which were then fixed at 1-120 hours
post-injection, then processed in parallel to insure comparable in situ
staining.

Examining these embryos (Fig.
4), we concluded that the injected mRNA diffused throughout the
injected teloblast and was readily passed on to the primary blast cell
progeny. The nGFP mRNA degraded more quickly within the blast cell clones than
within the teloblast and primary blast cells, however; n blast cells produced
after the injection stained intensely during the first 24 hours
post-injection, at which time they have not yet divided, but by 72 hours
post-injection the mRNA was barely detectable within the equivalent cell
clone. Comparisons with RDA injections indicate that this reflects mRNA
breakdown and not dilution (data not shown). At 120 hours post-injection, nGFP
mRNA was readily detected in the injected teloblast and supernumerary blast
cells and faintly in the most posterior segments, but not in any of the
anterior segmental progeny. Thus, the in situ signal results indicate the
perdurance of translatable transcripts in blast cell clones until ∼72
hours cl.ag. Finally, nGFP protein could be readily detected throughout the
germinal plate for as long as 145 hours post-injection, i.e. ∼3 days after
no nGFP mRNA could be detected (compare
Fig. 2D with
Fig. 4F). We conclude that the
anteroposterior gradient of nGFP reflects gradually increasing levels of nGFP
protein and decreasing levels of nGFP mRNA inherited by successive blast cells
from the parent teloblast, coupled with declining levels of residual nGFP
protein in older blast cells clones, from which the mRNA has been degraded.
Similar gradients of expressed protein were seen with all the synthetic mRNAs
used (data not shown).

Distribution and perdurance of injected mRNA. N teloblasts in stage 7
embryos were injected with nGFP mRNA. The embryos were fixed at times ranging
from 1-120 hours post-injection, then processed in parallel for in situ
hybridization. Arrowheads indicate the position of the first labeled clone,
where visible. (A-D) Animal pole views of embryos fixed 1-72 hours after
injection. The teloblast and relatively young blast cells (arrowhead in B)
stained intensely, but a significant decline in staining was apparent in blast
cell clones that had undergone their first mitosis (arrow in C,D). (E)
Prospective posterior view of an embryo fixed 96 hours after injection.
Although the teloblast still stained very intensely, the first labeled clone
(not visible in this view) was barely visible. (F) By 120 hours after
injection (side view), staining in the teloblast was clearly reduced, but
still clear. By contrast, nGFP mRNA was no longer detectable in the first
labeled clone under these conditions (arrowhead indicates the estimated
position of the first labeled clone; compare with
Fig. 2A,D). Scale bar: 100μ
m.

Cell lineage analysis by nGFP mRNA microinjection

We used the mRNA techniques described above to elucidate cell division
patterns in the N lineage. Knowing the complete lineage of the
Helobdella embryo is a long term goal, but here we focused on
differences between the ns and nf blast cell lineages, and resolving
uncertainties regarding the fates of the early progeny of the N
teloblasts.

Ganglionic primordia separate as the nf.p clone in one neuromere
delaminates from the ns.a clone in the next (fissure formation)
(Shain et al., 1998). To
identify differences between the nf and ns clones, we sought to compare the
lineages leading from the continuous columns of primary blast cells to the
separation of ganglionic primordia. Fissure formation occurs between∼
45-50 hours cl.ag. in Theromyzon rude
(Shain et al., 1998),
corresponding to ∼85-95 hours cl.ag. in the Austin strain of H.
robusta (Fig. 1).

For this purpose, tandem injections ∼90 minutes apart were used to
uniquely label single blast cell clones
(Zackson, 1982). Labeled
embryos were then examined by time-lapse confocal microscopy, beginning at 58
hours cl.ag., by which time the first labeled clone (an ns clone) contained
two or three cells (Fig. 5A).
To identify the limits within which photo-damage did not perturb development,
we compared the patterns of labeled nuclei in embryos subjected to various
illumination paradigms with those in equivalent embryos that were imaged only
at the end of the experiment. We found that sampling embryos at a 1 hour time
interval was sufficient to capture cell divisions and movements in the N
lineage during the period of interest, and that after a 12 hour observation
period, the pattern of labeled nuclei was as in an unirradiated sibling embryo
(Fig. 5A). These results
suggested that cell divisions had occurred normally during this period in the
imaged embryos. However, imaging periods greater than 12 hours often resulted
in arrested cell division, so to follow further events in the nf and ns
lineages, we undertook multiple overlapping time-lapse imaging experiments
covering 40-86 hours cl.ag. for the ns clone and 40-82 hours cl.ag. for the nf
clone.

Application of nGFP mRNA injections to analysis of the N lineage. (A) Time
series showing the anterior portion of an n bandlet; one ns blast cell was
uniquely labeled by tandem injections of the parent teloblast, first with nGFP
mRNA and then, after one blast cell was born, with RDA. Beginning 58 hours
after the first injection (I, 58-70), the uniquely labeled clone was imaged
hourly for 12 hours. During this time, the clone increased from two cells to
seven; a similar pattern of nuclei was observed in a sibling embryo imaged
only at 70 hours cl.ag. (II, 70). (B) Reconstruction of an embryo injected as
in A at 86 hours cl.ag. In the first (ns) labeled clone, superficial nuclei
are pseudocolored lavender and deep nuclei (2, 4, 8, 11, 12, 14) are red (8
and 12 are obscured by overlying nuclei). In the next (nf) clone, nuclei are
pseudocolored green. Those in more posterior clones appear white. The numbers
over the nuclei of the ns and nf clones correspond to cells in the lineages
trees in Fig. 6A and B,
respectively. (C) Three-dimensional reconstruction of a similar embryo with
the ns clone at 90 hours cl.ag. The ns clone (lavender) now has 18 nuclei; the
posterior nf clone (green) has 18 interphase nuclei and one in mitosis (broken
outline); the exact identities of these cells remain to be determined. In this
panel, the cytoplasmic RDA fluorescence is shown in red surrounding nuclei in
the nf and more posterior (white) clones, so that the elongating fissure
between prospective ganglionic primordia is visible (arrow); midline towards
left in B and C. Scale bar: 10 μm.

For this, the N teloblast was injected with nGFP mRNA at the beginning of
stage 7. Under this protocol, the first labeled clone was invariably the ns
clone contributing to the fourth segmental neuromere of the rostral ganglionic
mass (neuromere R4). Unfortunately, our observations ended ∼4 hours and
several cell divisions prior to the appearance of the transverse fissure
separating neuromeres R4 and M1 (Fig.
5B,C). The ns and nf blast cell clones had increased to 14 and 8
cells, respectively, by this time, including one dying cell in each clone
(revealed by nuclear morphology and cell fragmentation; data not shown;
Fig. 5B,
Fig. 6); apoptoses of unknown
lineage within the germinal bands had previously been reported
(Tsubokawa and Wedeen, 1999).
Three results of this study were as follows.

Diagram of the early ns (A) and nf (B) blast cell lineages (non-linear time
scale). Cell nomenclature is adapted from C. elegans as described
previously (Huang et al.,
2002). anterior, a; posterior, p; medial, m; lateral, l;
superficial, s; deep, d. The cl.ag. given for each division represents the
average of observations drawn from three to five specimens in each case.
Numbers correspond to the cells in Fig.
5B.

First, the lineages were consistent; the orientation and relative order of
mitoses was preserved within each clone, although the timing varied up to
several hours between embryos. This could reflect temperature differences
between experiments. Cell movements, judged by changes in the relative
positions of nuclei, were also stereotyped.

Second, many divisions exhibited stereotyped asymmetries, as judged by the
relative volumes of the nuclei of the daughter cells
(Fig. 7). Asymmetric divisions
of the nf and ns blast cells have been described qualitatively
(Bissen and Weisblat, 1989;
Song et al., 2002). Here, we
measured the nuclear volume ratios for the daughter cell pairs of 26 ns and 31
nf divisions. There was no overlap between the ratios for ns and nf divisions
and little variation within each class of n blast cells
(Fig. 7B), despite the fact
that the exact clonal age and position of the dividing cells varied
significantly between embryos. We conclude that the differentially asymmetric
ns and nf divisions reflect inherent differences between the two blast cell
types. Asymmetric cell divisions occurred throughout the regions of the ns and
nf lineages studied here. After each asymmetric division, the smaller daughter
cell (as judged by nuclear volume) invariably had a longer cell cycle than its
sister, but there was not a strict correlation between nuclear volume and cell
cycle duration overall (data not shown).

Third, the ns clone underwent more divisions and fewer cell rearrangements
than nf during the period under investigation. Cell divisions within the nf
clone were largely confined to the successive anterior daughter cells, as if
this cell were serving as a neuroblast, and nf.p did not divide at all. By
contrast, cell divisions occurred throughout the ns sublineage, which thus
contained roughly twice as many cells as the nf clone by the end of the period
(Fig. 6). Similar results were
obtained for the H. robusta strain isolated from Sacramento
(California) (F. Z. Huang, personal communication). These experiments focused
on the ns and nf lineages in neuromere R4, but similarly structured clones
occur throughout the labeled bandlet, indicating that other ns and nf clones
undergo similar patterns of early divisions.

Non-standard N teloblast progeny

The N teloblasts also contribute two sets of non-segmental cells to the
anterior micromere cap (Smith and
Weisblat, 1994). One is a clone of squamous epithelial cells
derived from micromere n′, which arises from the N teloblast after it
has already made three divisions (Sandig
and Dohle, 1988; Bissen and
Weisblat, 1989) (Fig.
8A). The other is a small domain of apparently columnar epithelial
cells derived from one or more of the first three N teloblast progeny. To
enable comparisons with lineage maps in other spiralians, we have resolved
uncertainties regarding the fates of the cells produced prior to micromere
n′.

Early progeny of the N teloblast. (A) Lineage diagram showing divisions of
the N teloblast. After its birth (stage 6a, ∼22 hours AZD), the N
teloblast divides every 90 minutes, generating a column of progeny that mostly
follow the ns and nf (segmental founder cell) fates in exact alternation. But
the fourth cell (n′) contributes exclusively to the provisional
epithelium, and the fates of cells born prior to n′ were not known with
certainty. (B) Pseudocolored 3D reconstruction of an nGFP-labeled n bandlet,
48 hours after the N teloblast was injected. The clone of the anteriormost
cell contains four nuclei of equal size (yellow), unlike either nf or ns, so
this is designated n°. The three cells posterior to this have each divided
once, in manner characteristic of the nf (green) or ns (red) clones. The
nucleus in the sixth position (broken outline) has entered mitosis before the
primary blast cell just ahead of it, also indicative of the nf (green) and ns
(red) fates. By this time, the n′ micromere clone contains six cells
(blue), superficial to the bandlet. (C) Equivalent view of an older embryo, in
which the N teloblast was re-injected with RDA after the birth of n° (all
nuclei are shown as yellow). By cl.ag. 96 hours, the n° clone comprises
70-80 nuclei, distinguished by their smaller size and less intense
fluorescence. Nuclei of the RDA labeled nf and ns blast cells are larger,
brighter and surrounded by RDA fluorescence (red). The n° clone is now
flanked by cells derived from the anteriormost nf clone (arrows). Fissures
(arrowheads) have formed between the posterior edges of the nf clones and the
anterior edges of the adjacent ns clones. (D) Ventral view of the
subesophageal ganglion and presegmental tissue of an embryo (∼146 hours
AZD) in which the left N teloblast was injected with RDA shortly before the
birth of its first ns blast cell, i.e. after it had already produced cell
n° and the first nf blast cell. The embryo was counterstained with Hoechst
33258 (green). Broken outlines indicate the edges of the four neuromeres
(R1-R4) in the subesophageal ganglion. Neuromere R1 contains the same
complement of labeled neurons as neuromeres R2-R4, even though its anterior
edge is unlabeled. (E) Pseudocolored 3D representation of the RDA-labeled
cells in an embryo equivalent to that shown in D. (F-H) Pseudocolored images
(processed as in C) of an embryo in which the N teloblast was injected with
RDA after the birth of n° (i.e. one cell cycle earlier than in D and E).
The N teloblast had also been injected with nGFP mRNA prior to the birth of
n°. As a result, the n° clone is labeled with nGFP only, and the first
RDA-labeled clone is descended from the first nf cell. (F) Ventral view,
comparable with E, showing only the RDA labeled cells; broken outline
surrounds the first nf clone. (G) The same image as in F, with the addition of
the n°-derived nuclei (yellow). (H) Side view (ventral to left) of the
image shown in G. Scale bar: 10 μm.

Tandem injections were used to label uniquely the first cell produced by
the N teloblast. We found that this cell follows neither the nf nor the ns
fate, but instead contributes exclusively to the set of columnar epithelial
cells. This cell (now designated n°) proliferates via equal and relatively
rapid cell divisions. Its clone comprises four nuclei at cl.ag. 48 hours
(Fig. 8B) and more than 80
cells by cl.ag. 96 hours (Fig.
8C). The second and third cells produced by the N teloblast follow
the nf and ns fates, respectively, as judged by the timing and asymmetries of
their first divisions (Fig.
8B), and the distribution of their progeny
(Fig. 8C). As described
previously, the fourth daughter of the N teloblast is micromere n′, then
the N teloblast reverts to the production of alternating nf and ns blast
cells, beginning with nf.

Finding that the anteriormost n blast cell is of the nf class is
paradoxical because in standard neuromeres, the ns clone lies anterior to the
nf clone (Fig. 1)
(Bissen and Weisblat, 1987).
The paradox is resolved by the realization that the anterior neuromere of the
subesophageal ganglion (R1) contains extra neurons compared with other
neuromeres (Fig. 8D). These
extra neurons arise from the nf clone (Fig.
8E,F), which could explain observations that the subesophageal
ganglion contains more (N-derived) serotonergic neurons than expected from
four fused standard neuromeres (Lent et
al., 1991). Although we propose that it forms the anteroventral
adhesive organ (Smith and Weisblat,
1994), the definitive fate of the n° clone remains to be
determined. By 120 hours cl.ag., the n°-derived cells lie immediately
ventral to the first nf clone (Fig.
8G,H).

Polarity and asymmetry of blast cell divisions

To examine the regulation of the asymmetric n blast cell divisions, we
injected left N teloblasts with tau::GFP mRNA, then followed spindle dynamics
in 26 ns and 18 nf divisions.

Mitosis took about 30 minutes for both nf and ns blast cells. No
differences were observed until late in mitosis; in both ns and nf, spindle
assembly was evidenced by increasing fluorescence intensity at the centrosomes
(Fig. 9A, 00-04 minutes;
Fig. 9B, 00-05 minutes).
Centrosomes in the n blast cells had separated at several hours before the
onset of mitosis (Fig. 9C), and
usually retained an obliquely transverse orientation with respect to the AP
axis of the n bandlet as cells rounded up for mitosis
(Fig. 9A, 08 minutes;
Fig. 9B, 05 minutes). In three
of the divisions (1 nf and 2 ns), the centrosomes were already in an
anteroposterior orientation prior to the onset of mitosis. No asymmetries were
evident as the spindles began to rotate following nuclear breakdown
(Fig. 9A, 12-20 minutes;
Fig. 9B, 05-11 minutes).

Spindle rotation took 3-5 minutes and most spindles (22/26 ns and 16/18 nf)
rotated counterclockwise, in contrast to the random directionality of spindle
rotation in cell P0 of C. elegans
(Hyman, 1989). The blast cell
spindle rotations proceeded without apparent hesitation or reversal of the
spindle rotation, in apparent contrast to the jerky spindle rotation in cell
P0 of C. elegans, but this could result from the lower
sampling rate required in our fluorescence imaging experiments to minimize
photodamage. In several specimens, the spindle underwent back and forth
movements between the anterior and posterior cortices after rotation
(Fig. 9A, 20-24 minutes), as if
there were competing pulling forces from the anterior and posterior cortices,
as seen in C. elegans P0 cell divisions
(Grill et. al., 2001).

Differences between nf and ns blast cells appeared late in mitosis.
Cytokinesis occurred with the spindles displaced towards the posterior end of
the cell, with the result that the cleavage furrow was also shifted
posteriorly. The posterior shift of the spindle was correlated with a
diminution in fluorescence of the posterior aster relative to the anterior
aster in both cell types. The intensity difference between the anterior and
posterior poles was not observed until after the completion of spindle
rotation. Though we could not stain for DNA in these experiments, we estimate
that the change in the posterior spindle pole occurred at the onset of
anaphase. The posterior displacement of the furrow was greater in nf than in
ns (Fig. 9C), as was the
relative diminution of the posterior spindle pole fluorescence
(Fig. 9B, 22-24 minutes).
Within the nf blast cells, the spindle midbody was displaced towards the
posterior centrosome, which further increased the extent of the mitotic
asymmetry (Fig. 9C, 10
minutes). This difference between nf and ns correlates with the differences in
the nuclear volume ratios of their respective progeny
(Fig. 7).

Discussion

mRNA injections in Helobdella

Our results demonstrate the utility of routine mRNA injections for
expressing cytoplasmic and nuclear localized versions of GFP and related
fluorescent proteins, tau::GFP, and lacZ at readily detectable levels
in Helobdella embryos without perturbing normal development. In situ
hybridization revealed that the injected mRNAs are distributed throughout the
cytoplasm of the injected teloblast and its blast cell progeny. Consistent
with this, expressed proteins were seen in all progeny, in contrast to the
mosaic expression seen when DNA is injected into teloblasts (data not shown)
(Pilon and Weisblat, 1997).
Some RNA injections give mosaic expression in Xenopus and
Danio embryos (Sive et al.,
2000; Geldmacher-Voss et al.,
2003). We speculate that the slower cell divisions in leech may
give time for the injected RNA to spread prior to cytokinesis.

Injected mRNA is degraded more quickly in the blast cells than in the
teloblast, and more quickly within the germinal bands than in the bandlets.
Previous work has shown that blast cells accumulate transcripts more rapidly
than teloblasts (Bissen and Weisblat,
1991) and there is a dramatic prolongation of the cell cycle in
blast cells relative to teloblasts
(Zackson, 1984;
Bissen and Weisblat, 1989).
These features support the idea that the teloblast-to-blast cell transition in
leech is analogous to the `mid-blastula transition' in Xenopus and
Drosophila (reviewed by Yasuda
and Schubiger, 1992).

For all the mRNAs tested, we observed graded expression of the encoded
protein; the first blast cells born after injection contain low levels of the
protein and those born later contain progressively higher levels. No such
gradient is observed when passive tracers are used. From the in situ results
and the stem cell mode by which blast cells arise, we conclude that: (1)
clones founded by blast cells born immediately after the injection contain
only the relatively low levels of protein expressed before the mRNA they
inherit from the teloblast is degraded; and (2) blast cells born at
progressively later times post-injection inherit not only the mRNA, but also
inherit increasing amounts of protein expressed in the teloblast, in which the
injected message is relatively stable.

Whatever the mechanism by which they are formed, the gradients of protein
expression driven by injection of synthetic mRNAs means that dose effects of
mutant or ectopic regulatory and signaling proteins from such injections can
be determined simply by examining their effects on blast cells born at
different times and by comparing their effects on teloblast divisions at
various times after injection (S.O.Z. and D.A.W., unpublished). Finally, we
have demonstrated the efficacy and specificity of AS MO knockdown as a means
of modulating the expression of injected mRNAs. Together, these techniques
provide a powerful tool for functional analysis of gene function in an animal
for which standard genetic approaches are not available.

Application of the mRNA injection technique to analysis of the N
teloblast lineage

In Helobdella, most ganglionic neurons arise from the N teloblasts
via two distinct classes of blast cells, ns and nf, that arise in exact
alternation. Homologous lineages have been described in both lumbricid and
tubificid oligochaetes (Storey,
1989; Arai et al.,
2001). Therefore, this grandparental mode of stem cell division is
almost certainly ancestral to clitellate annelids. To further understand this
process, we have applied the mRNA injection technique in three ways.

We used nGFP lineage tracer to extend our knowledge of the nf and ns blast
cell lineages, and to ascertain the fates of the initial `non-standard' early
progeny of the N teloblasts. We find that the nf and ns lineages differ from
one another in terms of timing, orientation and asymmetry of mitoses.
Moreover, the nf lineage differs from ns in that the anterior cell at each of
the first four divisions shows a markedly shorter cell cycle time than its
posterior sister cell. As a result, the nf lineage shows a stem cell-like
pattern. Similar division patterns, but with a more rapid time course, have
been seen for a genetically distinct strain of H. robusta (F. Z.
Huang, personal communication). Regarding the early progeny of the N
teloblast, we find that the first cell born from the N teloblast undergoes
symmetric and relatively rapid divisions. This cell, which we designate
n°, generates a clone with no obvious homology to either nf or ns, and is
predicted to contribute to the adhesive organ. The second cell born from the N
teloblast gives rise to an nf-like clone. We predict that it contributes
`extra' N teloblast derivatives primarily to the most rostral of the segmental
neuromeres (R1) in the subesophageal ganglion.

Using the localized nGFP fluorescence, we quantified the nuclear volumes of
the sister cells resulting from nf and ns divisions. The volume ratios
(Vnf.p/Vnf.a and Vns.p/Vns.a)
varied only about 10% internally, and value for the nf progeny is less than
half that for the ns progeny. These results indicate that the asymmetric
divisions of nf and ns cell are under tight, differential control. Asymmetric
cell divisions are a prominent feature of the stereotyped division patterns
throughout the segmental and non-segmental tissues of the Helobdella
embryo (Zackson, 1984;
Shankland, 1987;
Huang et al., 2002); we find
that later divisions within the ns and nf blast cell lineages are also
asymmetric. Whether such asymmetries are essential for establishing fate
differences or merely a reflection of those differences remains to be
determined. The most obvious way in which the stereotyped asymmetry (and/or
orientation) of cell divisions could specify different fates for the progeny
is by segregating asymmetrically localized fate determinants, as in the
unequal first cleavage of the Helobdella embryo
(Whitman, 1878;
Astrow et al., 1987). Well
studied examples include unequal division of the C. elegans zygote
(Gotta and Ahringer, 2001) and
Drosophila neuroblasts (Knoblich,
2001), as well as the asymmetric division of the C.
elegans EMS cell that is induced by contact with blastomere P2
(Goldstein, 1995). Another
effect of asymmetric cell divisions could be to position the progeny with
respect to inductive influences of other cells, as seen in the EMS lineage of
C. elegans (Goldstein,
1995). A third means by which an asymmetric cell division could
influence cell fates would be by affecting the cell cycle duration and
composition of the progeny. For example, the smaller posterior progeny of the
n blast cells (nf.p and ns.p) have longer cell cycles than their siblings and
the homologous cells in a congenic species, H. triserialis, are the
first cells in their lineage to go through a measurable G1 phase
(Bissen and Weisblat, 1989). It
seems likely that, in addition to accumulating `housekeeping' transcripts and
proteins required to progress through the cell cycle, these cells would also
makes different types of quantities of developmental regulatory gene products
as well. To our knowledge, no definitive example of this third possibility has
been described.

To pursue the mechanisms by which the asymmetric n blast cell divisions are
controlled, we used tau::GFP fluorescence to follow n blast cell spindle
dynamics. Centrosomes in the blast cells have separated well prior to the
onset of mitosis. Spindles in the left n bandlet almost always rotate in a
counterclockwise direction. The spindle is initially positioned symmetrically
within the cell; the asters appear of equal size and fluorescence intensity
until after rotation. Asymmetry emerges during late metaphase or anaphase,
when the spindle moves posteriorly and the posterior aster undergoes a
dramatic reduction in size/fluorescence intensity. We interpret this to mean
that the astral microtubules of the anterior spindle pole are longer and/or
more stable than those of the posterior pole. Finally, at least in the nf
blast cells, a further degree of asymmetry is contributed by the posterior
shift of the spindle midbody relative to the spindle poles.

In C. elegans, the asymmetric division of the zygote is driven by
a posterior shift in the mitotic spindle
(Albertson, 1984). By contrast,
it has been reported that the asymmetric divisions of Drosophila
neuroblasts involve a basal shift of the spindle midbody relative to the
spindle poles (Kaltschmidt et. al.,
2000). Our findings show that these processes can occur together
in cells, and that they can be regulated differentially to generate the
distinct asymmetries of the alternating nf and ns blast cells in
Helobdella.

Acknowledgments

We are indebted to Yanzhen Cui and Richard Harland for providing plasmids
and for helpful discussions, which are crucial to the success of this work. We
thank Francoise Huang, Sara Agee, Shirley Bissen, Marty Shankland and members
of our laboratory for technical assistance and discussions; and Holly Aaron
(Berkeley Molecular Imaging Center) and Steve Ruzin (CNR Biological Imaging
Center) for assistance and training in confocal microscopy. This work was
supported by NIH grant RO1 GM60240 to D.A.W. and by UC Regents Fellowship to
S.O.Z.

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